Notch signaling pathways are important for several crucial decisions during lymphocyte development. The basic helix-loop-helix E2A transcription factors (E12/E47) are also essential for the development for both B and T lymphocytes. Our recent findings suggest that E2A transcription factors may be downstream effectors of Notch signaling for lymphocyte development. Specifically, we have shown that signaling through Notch modulates the turnover of E2A proteins, which requires the phosphorylation of these proteins by p42/p44 MAP kinases. Expression of activated Notch l enhanced the association of E47 with the SCFSkp2 E3 ubiquitin ligase and its ubiquitination. Notch-induced degradation of E2A proteins occurred in B but not T cells studied. However, this correlated with the level of MAP kinase activity in these cells. Therefore, differences in the level of MAP kinase activity may be an effective regulatory mechanism to control E2A turnover. In this proposal, we first hypothesize that accelerated E2A degradation by Notch signals may play an important role in Notch-mediated T versus B lineage choice by blocking B cell development in the thymus. We then propose that Notch signals may be responsible for reducing E2A levels in T cells once functional pre-TCR and TCR are formed, and lower E2A levels are necessary for T cells to go through proper selections during maturation. These hypotheses will be tested by measuring E 2A degradation in progenitor B cells and in T cells upon activation of MAP kinases. Furthermore, mutations will be introduced into the E2A genome to encode E2A proteins resistant to Notch induced degradation. The knock-in mice will be used to determine if expression of the mutant protein allows B cell formation in the thymus, renders resistance to Notch-mediated inhibition of B cell development in the bone mar row, and impacts on T cell selection, as well as positive and negative selection. Finally, we propose to understand how Notch signals induce the ubiquitination and degradation of E2A proteins. Perhaps, Notch signals facilitate expression of unknown genes involved in the ubiquitination of E2A proteins, which will be identified by examination of proteins associated with the ubiquitination machinery of E2A proteins. In summary, studies outlined here expand our investigation on a novel function of Notch signaling, i.e., induction of ubiquitin-mediated E2A degradation, and evaluate the biological significance of this function in the context of lymphocyte development.